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Results

We constructed and tested a bipartite GBT vector with Gal4-VP16 as the primary gene
trap reporter. Our vector also contains a UAS:eGFP cassette for direct detection of
gene trap events by fluorescence. To confirm gene trap events, we generated a UAS:mRFP
tester line. We screened 270 potential founders and established 41 gene trap lines.
Three of our gene trap alleles display homozygous lethal phenotypes ranging from embryonic
to late larval: nsftpl6, atp1a3atpl10 and flrtpl19. Our gene trap cassette is flanked by direct loxP sites, which enabled us to successfully
revert nsftpl6, atp1a3atpl10 and flrtpl19 gene trap alleles by injection of Cre mRNA. The UAS:eGFP cassette is flanked by direct
FRT sites. It can be readily removed by injection of Flp mRNA for use of our gene
trap alleles with other tissue-specific GFP-marked lines. The Gal4-VP16 component
of our vector provides two important advantages over other GBT vectors. The first
is increased sensitivity, which enabled us to detect previously unnoticed expression
of nsf in the pancreas. The second advantage is that all our gene trap lines, including
integrations into non-essential genes, can be used as highly specific Gal4 drivers
for expression of other transgenes under the control of Gal4 UAS.

Conclusions

The Gal4-containing bipartite Gene Breaking Transposon vector presented here retains
high specificity for integrations into genes, high mutagenicity and revertibility
by Cre. These features, together with utility as highly specific Gal4 drivers, make
gene trap mutants presented here especially useful to the research community.

Keywords:

Background

Amenability to mutagenesis combined with optical transparency of externally developing
embryos and large clutch size make zebrafish an excellent model system for developmental
genetics [1]. Two large-scale ethylnitrosourea (ENU) mutagenesis screens have clearly established
that all aspects of zebrafish development can be analyzed by forward genetics [2-4]. Indeed, these large-scale screens continue to be followed up by more specialized
screens for defects in specific biological pathways (reviewed in [5]). High efficiency and random nature of chemical mutagenesis enables generation of
multiple alleles of variable strength, as recently reported for tbx2b[6,7]. Constant improvements in both mapping resources and fidelity of the assembly of
the zebrafish genome have enabled identification of a significant subset of genes
affected by chemically-induced mutations. Nonetheless, some chemical mutants of exceptional
biological interest remain to be cloned or confirmed. A classic example is the hemangioblast
mutant cloche, for which only a candidate gene has been reported to date [8,9]. Other examples are still-uncloned cocaine addiction mutants dumbfish, jumpy and goody-two-shoes[10]. Thus, while chemical mutagenesis can readily generate mutants in biological pathways
of interest, it does not always lead to molecular identification of the affected genes.

Insertional mutagenesis is not as random as chemical mutagenesis and does not possess
as high efficiency. However, these deficiencies are offset by more straightforward
identification of affected genes using the insertional mutagen as a molecular tag.
Furthermore, it is possible to use fluorescent reporters to monitor the expression
of mutated genes as well as design the insertional mutagen for additional utility.
The only insertional mutagen used to date in large scale in zebrafish is the pseudotyped
retrovirus. The virus has been used in two complimentary approaches. The first was
to mutate genes required for embryonic development, leading to identification of over
330 such genes by a single laboratory [11-13]. The second approach was to analyze the regulatory landscape of the zebrafish genome
through enhancer trapping [14]. Albeit successful, retrovirus as an insertional mutagenesis vector has several limitations.
First, production and handling of viral particles requires special expertise and facilities.
Second, modifications such as addition of gene trap components may result in lower
virus titers and require significant optimization of production procedures [15]. Finally, the only retroviral approach that produced fluorescent protein expression-tagged
integration events -the enhancer trap- did not produce a significant number of loss-of-function
alleles [14].

Success of transposon-based mutagenesis in Drosophila (reviewed in [16]) prompted investigation of the activity of different transposons – Tc1, Sleeping Beauty and Tol2- in the zebrafish [17-19]. In contrast to the retrovirus, transposable elements do not possess the machinery
to deliver exogenous DNA into the nucleus, which results in somewhat lower rates of
integration into the genome and subsequent germline transmission of transposition
events. Transposon-based insertional mutagenesis vectors used in zebrafish fall under
the general categories of enhancer trap, 5’ gene trap and 3’ gene trap, and include
fluorescent reporters to detect “trapping” events. Enhancer and 5’ gene trap vector
integrations reveal the expression profile of the tagged locus and are ideal for the
optically transparent, externally developing zebrafish embryos. The drawback of enhancer
trap vectors is that they usually are not mutagenic and only induce mutations by integrations
into exons or other essential sequences of genes (reviewed in [16]). As expected, transposon-based enhancer trap integrations did not result in overt
embryonic phenotypes in zebrafish [20,21]. Initial zebrafish gene trap vectors suffered from poor mutagenicity as well [22]. Two reasons may underlie this lack of mutagenicity. First, the vector used in these
studies was later found to also function as an enhancer trap [23]. Second, when integration did occur into a gene, the splice acceptor and polyadenylation/transcriptional
termination sequences were leaky, allowing for read-through transcription and splicing
[22,24]. This leakiness may be partly attributed to use of rabbit β-globin splice acceptor
(SA) [22], as there appear to be significant differences between mammalian and fish splice
sites [25]. To reduce this read-through transcription and splicing, Sivasubbu and colleagues
used fish-derived SA and transcriptional termination/polyadenylation (p(A)) sequences.
Integrations into genes were selected using the 3’ gene trap paradigm and enabled
identification of the first transposon-induced phenotypic mutation in zebrafish, leading
to adaptation of the term “Gene Breaking Transposon” (GBT) [26]. Notably, GBTs are capable of inducing mutations by integration into introns of genes.
This makes GBT-induced mutations reversible by removing the SA/p(A) components.

The next step was to develop a true 5’ gene trap vector by flanking it with a fish-derived
and potentially mutagenic SA/p(A) sequences. To make selection for integration into
protein-coding genes more stringent, the AUG translation initiation codon of the fluorescent
reporter was removed. The 5’ gene trap cassette was flanked by loxP sites, leading
to generation of first revertible mutants in zebrafish [27,28]. An alternative mutagenesis strategy relying on integration of trap vectors into
exons or other important sequences has also been proposed [29,30]. In parallel, Gal4-based transcriptional activators were being adapted for insertional
mutagenesis [23,31,32]. The main advantage of Gal4-based transcriptional activators is that gene- or enhancer-trap
lines can be used as drivers for expression of other transgenes including fluorescent
reporters, toxin genes, calcium sensors and optically activated channel proteins,
in a tissue-specific manner [23,31,33-36].

In this study, we provide proof-of-principle demonstration that a gene breaking transposon
can be equipped with Gal4-VP16, resulting in sensitive detection of weak gene expression.
Genes involved in a variety of cellular functions, from transcription to secretion,
were mutated using our Gal4-based vector. The modified gene trap is highly mutagenic
at molecular and phenotypic levels, resulting in isolation of two embryonic lethal
and one post-embryonic lethal mutations which are revertible by Cre-mediated recombination.

Results

Gene trap vector design and features

Our GBT-B1 (for Gene Breaking Transposon- Bipartite 1) vector is composed of several components that in concert ensure efficient
mutagenesis, evaluation of the trapped gene’s expression profile and enable manipulation
of resulting mutant alleles. It is based on the GBT-R15 vector (Figure 1A) [27,28], with AUG-less mRFP (^mRFP) replaced by AUG-less Gal4-VP16 (^Gal4-VP16) (Figure 1B). For direct detection of gene trap events, GBT-B1 vector also contains a UAS:eGFP
cassette [23,34,36]. In addition, the vector has FRT, loxP and I-SceI meganuclease sites to facilitate
manipulation of insertional alleles. The loxP and I-SceI sites, originally present
in the parental GBT-R15 vector [27,28], flank Gal4-VP16 and UAS:eGFP sequences. The two loxP sites are in direct orientation
and therefore permit excision of the gene trap leading to reversion of the mutations,
as demonstrated for GBT-R15 mutations in gabbr1b and tnnt2 genes [27,28].

In Drosophila, P-element integrations can be converted into deletions (deficiencies)
by imprecise excision of the transposon [38,39] (reviewed in [40]). As Tol2 transposon does not appear to be prone to imprecise excision, we flanked
our gene trap cassette with two inverted I-SceI meganuclease sites. While I-SceI is
mainly used to facilitate transgenesis in zebrafish, it is used to study DNA repair
pathways in other systems [41-44]. We anticipate that double strand breaks induced by I-SceI meganuclease may be repaired
by error-prone repair mechanisms, leading to generation of local deletions large enough
to remove one or more coding exons of the mutated gene.

To test if our gene trap vector containing Gal4-VP16, UAS:eGFP and additional features
retained the specificity of GBR-R15, we injected this vector into 1-cell zebrafish
embryos with and without Tol2 transposase mRNA. Without Tol2 transposase mRNA, there
was no expression of eGFP (Figure 1C). When Tol2 transposase was included, almost all embryos had GFP-positive cells,
with 20-30% expressing GFP quite highly and/or in tissues of different embryonic origin
(Figure 1D). We concluded that GBT-B1 is unable to express eGFP unless integrated into the
genome, and therefore may have sufficient fidelity to function as a gene trap.

Gal4-VP16 has been shown to be toxic when overexpressed, and an attenuated version
of the activator, Gal4-FF, has been shown to activate expression of UAS-controlled
transgenes in zebrafish [32,45-47]. We therefore constructed a second gene trap vector, named GBT-B2, which contains
Gal4-FF in place of Gal4-VP16. When injected into zebrafish embryos, GBT-B2 produced
significantly lower level of eGFP expression, suggesting that Gal4-FF is a significantly
less potent transcriptional activator than Gal4-Vp16 (Additional file 1: Figure S1). We screened 58 F0 fish injected with GBT-B2 and failed to recover any
gene traps. Gal4-FF may be too weak a transcriptional activator to function in the
context of a highly stringent gene trap requiring a translational fusion with the
N terminus of the protein encoded by the mutated gene.

UAS:mRFP tester lines

Integration of GBT-B1 into an intron of a protein-coding gene in sense orientation
is expected to result in a fusion transcript between the 5’ end of the IMG (Insertionally Mutated Gene) mRNA and Gal4-VP16 (Figure 2). If the reading frames of the upstream exon of the IMG and Gal4-VP16 match, a fusion
protein composed of the N-terminal portion of the protein encoded by IMG and Gal4-VP16
will be translated. For a gene trap to be detected, this fusion protein has to enter
the nucleus, bind the 14XUAS and activate eGFP expression. However, eGFP expression
may also result from an enhancer trap event if the minimal promoter in front of eGFP
falls under the control of a nearby enhancer (Additional file 2: Figure S2, see also [48]). It is critical to distinguish between gene- and enhancer-trap events. Gene traps
represent integrations into genes that disrupt their expression, while enhancer trap
events are unlikely to be integrations into genes and consequently unlikely to result
in any loss-of-function phenotypes. To distinguish between these two classes of events,
we have generated two 14XUAS:mRFP transgenic lines. In case of a bona fide gene trap event, the IMG-Gal4-VP16 fusion protein will activate the expression of
UAS-driven mRFP in trans. In case of an enhancer trap, eGFP is produced in the absence of Gal4-VP16 and UAS:mRFP
will not be activated.

Our first gene trap tester line, Tg(miniTol2/14XUAS:mRFP), was established based on weak leaky expression of mRFP in the nervous system. Leaky
mRFP expression in the absence of transactivation complicated gene trap screening,
and we no longer use this transgenic line. Our second UAS:mRFP tester line is Tg(miniTol2/14XUAS:mRFP, γCry:GFP)tpl2 (Tg(UAS:mRFP)tpl2 for brevity hereafter), marked by a lens-specific GFP cassette from [19]. To reduce the possibility of mixing up putative founder or F1 gene trap fish with
Tg(UAS:mRFP)tpl2 tester fish, we bred Tg(UAS:mRFP)tpl2 onto a homozygous brass background and inject gene traps into fish with wild type pigmentation pattern.

Screening strategies

The gene trap vector DNA was mixed with Tol2 transposase mRNA and injected into the
yolk of 1-cell zebrafish embryos. Injected embryos were screened for GFP expression
at 3 days post fertilization (dpf). Approximately 30% of embryos with the brightest
GFP expression (Figure 1D) were selected and raised to establish an F0 pool for screening. The pilot gene
trap screen was carried out in two stages. In the first stage, the F0 fish were incrossed,
and all GFP-positive embryos were raised. GFP-positive fish were then crossed to Tg(miniTol2/14XUAS:mRFP) or Tg(UAS:mRFP)tpl2 to distinguish between gene trap events (mRFP-positive) and enhancer trap events
(mRFP-negative). Seventy fish were screened and 13 gene trap lines were established
from this work. We have also discovered at least 10 enhancer trap events. Enhancer
traps were discarded with the exception of Et(GBT-B1)tpl1, which displayed a highly specific vascular expression pattern [48]. In the second stage of the screen, GBT-B1-injected F0 fish were screened by crossing
to the Tg(UAS:mRFP)tpl2 line. We screened two hundred putative F0 fish and recovered 28 gene trap events
from the second stage of the screen. Altogether, we screened 270 putative F0 fish
and recovered 41 gene trap lines with diverse expression patterns (Figure 3).

Figure 3.Expression patterns recovered from the gene trap screen. Each gene trap line is represented by two images: brightfield and fluorescence. Most
of the lines are represented by expression pattern of the fluorescent reporter observed
in 3dpf embryos, except for those lines were reporter’s expression pattern is best
visible in embryos at earlier stages of development. The latter include lines tpl8, tpl11, tpl19, tpl30, tpl34 and tpl39 for which embryos were imaged at 1 dpf, as well as lines tpl4, tpl6, tpl16, tpl21, tpl22, tpl25, tpl27, tpl28, tpl40 and tpl42 that are represented by 2 dpf embryos.

Identification of insertionally mutated genes (IMGs)

To estimate the number of integrations present in F2 fish, we have performed Southern
hybridization analysis with an eGFP probe on a pool of 20 GFP positive and a pool
of 20 GFP negative embryos from 13 different lines. The analysis revealed that the
number of transposon insertions varied from 1 to 12 (data not shown) among different
lines, and in most of them there was only a single integration linked to GFP.

Two complementary strategies were used to identify insertionally mutated genes: inverse
PCR (iPCR) and 5’ RACE. iPCR is the preferred method for mapping IMGs as it permits
identification of the exact genomic position of the gene trap integration. In contrast,
5’RACE only identifies the 5’ exon(s) of the trapped gene, but does not reveal the
exact genomic position of gene trap integration, which makes it challenging to design
genotyping primers.

iPCR analysis led to successful identification of IMGs in 21 of the lines while for
the remaining 20 lines iPCR results were inconclusive, yielding either repetitive
sequences that have multiple matches in the zebrafish genome, sequences that mapped
to short contigs devoid of protein coding genes, or sequences that did not map onto
current assembly of the zebrafish genome. In some cases, inverse PCR failed to produce
any bands at all. For the majority of candidate IMGs (cIMGs) identified by iPCR, the
presence of a fusion mRNA in GFP-positive but not GFP-negative embryos was also confirmed
by conducting RT-PCR with a cIMG’s exonic primer upstream of the integration and a
reverse Gal4 primer. Fusion mRNAs were then sequenced to verify continuity of the
reading frame between the upstream IMG exon and Gal4-VP16.

To identify additional IMGs, we performed 5’-RACE on four gene trap lines that failed
iPCR mapping: tpl3, tpl8, tpl9 and tpl15. Similarly to iPCR, 5’ RACE was performed on RNA from batches of 20 GFP positive
embryos following a published protocol [28]. Linkage of a given 5’ RACE product to GFP expression was confirmed by RT-PCR on
mRNA from batches of 20 GFP-positive and GFP-negative embryos collected independently
of the 5’RACE procedure. We successfully identified cIMGs in all four lines. For gene
trap line nudCtpl3, exons upstream of the gene trap integration site are missing from Zv9 zebrafish
genome assembly, which explains why inverse PCR failed to map this integration. In
gene trap line zfp36l1atpl8, the transposon integrated into a relatively short (4 kb) intron. We were able to
map the exact position of the gene trap integration by carrying out PCR using exon
primers in combination with Tol2 inverted repeat primers. We were unable to determine
the exact position of the gene trap integration in lines nfe2l1tpl9 and eef1a1l1tpl15.

Altogether, we established 41 gene trap lines and successfully identified IMGs linked
to GFP expression in 25 of them (Table 1, Figure 4A). In 20 cases, gene trap integration has occurred into an intron following an exon
ending in phase 0 with respect to the gene’s reading frame. This is the expected scenario,
as the reading frame of Gal4-VP16 in our gene trap vector is in phase 0 with respect
to the splice site. Among the five cases not conforming to this expected scenario,
four are integrations into exons. In gene trap lines st6GalNAc5tpl5, dkey-9i23.6tpl13 and cyp26C1tpl24, the transposon integration occurred into exons immediately following an exon in
phase 0. We did not perform RT-PCR on dkey-9i23.6tpl13, but in gene trap lines st6GalNAc5tpl5 and cyp26C1tpl24 RT-PCR yielded fusion transcripts consisting of the upstream exon and Gal4-VP16.
This occurs if splicing machinery skips the endogenous splice acceptor upstream of
the exon into which the gene trap integrated and used the gene trap splice acceptor
instead. The fourth gene trap line with an exonic gene trap integration linked to
GFP expression is fam46bbtpl18. In this case, the gene trap has integrated into the first exon of the gene, downstream
of the ATG but 14 base pairs upstream of exon/intron boundary. It is likely that another
linked gene trap integration we failed to identify may be responsible for the GFP
expression pattern. Nonetheless, fam46bbtpl18 can be considered a null mutant of fam46bb. The fifth non-canonical case of gene trap integration is presented by jam3btpl7 line. In this case, gene trap integration into the first intron of jam3b gene is linked to GFP expression, and expression in our gene trap line closely resembles
the published expression pattern of jam3b (see zfin.org). However, the first exon of jam3b ends in phase 1, which should prevent translation of Jam3b-Gal4VP16 fusion protein.
We confirmed this out-of-frame fusion transcript by RT-PCR. We also observed that
two gene trap integrations were linked to GFP expression by Southern hybridization
(data not shown), indicating that additional integrations on chromosome 21 may be
responsible for the gene trap expression pattern.

Figure 4.Characterization of GBT-B1 gene trap events at the molecular level. (A) Schematic illustration of molecularly characterized gene trap events. Gene trap integration
is indicated by an open triangle. Exons upstream of gene trap integration are shown
as black (coding exons) or grey (non-coding exons) boxes. Exons downstream of gene
trap integration are shown as open boxes. Integrations into jam3b and fam46bb are not shown because they do not form in-frame fusions with the upstream exon. (B) Levels of wild-type transcript present in homozygous gene trap mutants.

Our gene trap screen yielded a variety of expression patterns, ranging from fairly
ubiquitous to tissue-specific (Figure 3). Expression patterns of UAS:eGFP in cis and UAS:mRFP in trans matched very closely. Consistently with published observations [49], eGFP expression was quicker to appear in all gene trap lines, but mRFP expression
was more robust in later stages of development. Expression patterns of many of the
mutated genes have been previously described by others, and data has been deposited
to Zebrafish Information Network (zfin.org), including not spatially restricted expression
patterns noted for nudc (tpl3), stat5.1 (tpl4), eef1a1l1 (tpl15) and lasp1 (tpl20). Other expression patterns available through ZFIN reasonably closely match our observed
expression patterns with some notable exceptions. Several of the gene traps display
pronounced mRFP fluorescence in the yolk (tpl4, tpl9, tpl14, tpl21, tpl26, tpl35, tpl39), while gene expression in the yolk is not typically observed by in situ hybridization. This may be non-specific, or may reflect accumulation of fluorescent
protein from maternal contribution or from earlier gene expression in yolk syncytial
layer. A separate subset of gene traps displays mRFP expression in the notochord (tpl5, tpl17, tpl18, tpl31, tpl32) at 3 dpf. Similarly to yolk, notochord is not a prominent expression domain when
gene expression at 3 dpf is analyzed by in situ hybridization (zfin.org and [50]). mRFP expression in the notochord may be a remnant of earlier expression, or may
reflect non-specific background. A particularly instructive example is presented by
the baiap2l1atpl22 gene trap. By in situ hybridization, baiap2l1a is expressed in the periderm and notochord before but not after the 25-somite stage.
In 2-day embryo, baiap2l1a is expressed in the pronephric duct, in the general area of the pharynx and in the
brain (zfin.org and [50]). In contrast, mRFP expression in the skin and the notochord is observed through
3 dpf in baiap2l1atpl22 gene trap line, in addition to highly pronounced expression in the areas of kidney
tubules, pharynx and lower jaw (Figure 3). It has been noted that turnover of mRFP in vivo is very slow [49], which may explain the presence of UAS-driven mRFP but not baiap2l1a mRNA in the skin and the notochord at 3 dpf.

In the nsfatpl6 gene trap line, we observed expression of GFP throughout the developing nervous system
in a pattern largely consistent to the previously reported expression of nsfa[51,52]. However, the gene trap line nsfatpl6 also expresses GFP and RFP in the pancreas (Figure 3). To investigate if this pancreatic fluorescent protein expression corresponds to
endogenous expression of nsfa, we performed whole-mount in situ hybridization using a DIG-labeled riboprobe antisense to nsfa. Robust in situ hybridization signal in the nervous system was detectable after a short staining
period. Longer incubation yielded a clear hybridization signal in pancreas as well
(Additional file 3: Figure S3). This indicates that endogenous nsfa transcript is present in the pancreas. Relatively low abundance of nsfa transcript in the pancreas compared to the nervous system explains why it was missed
by previous studies in zebrafish. Importantly, nsf is known to be expressed in human pancreatic beta-cells [53].

While all of our Gal4 gene traps can be used as drivers to express other transgenes
in tissues expressing mRFP (Figure 3), lines displaying restricted neuronal expression are of particular interest given
presence of well-established Gal4/UAS based tools to modulate and detect neuronal
activity [32,35,54-57]. We therefore decided to test how closely expression of mRFP in trans corresponds to the endogenous expression of three genes expressed in overlapping
but different neuronal domains: ebf3, cyp26c1 and snap25b (Figure 5). We found that expression of mRFP largely corresponds to the expression of endogenous
gene, but in an incomplete and/or mosaic pattern. Incompleteness of mRFP expression
compared to the endogenous expression may in part be due to the delay in mRFP fluorescence
because of slow maturation of mRFP as well as the additional step of transcriptional
activation by Gal4-VP16. Mosaicism of mRFP expression compared to the expression of
endogenous gene is most likely due to partial silencing of Gal4 UAS. Thus, while BGT-B1
gene traps can be used as highly specific Gal4 drivers, additional steps should be
taken to ensure that the transgene of interest is indeed expressed in the cells that
are being targeted.

Assessment of mutagenicity at the molecular level

One of the key questions regarding any mutagenesis strategy is the ability to generate
complete loss-of-function (null) alleles. Reverse genetic strategies such as tilling,
zinc finger nucleases and TALEN nucleases in zebrafish and homologous recombination
in the mouse are designed to ensure physical disruption of exons or splice sites,
which greatly increases the probability of complete loss-of-function alleles. Generation
of loss-of-function alleles by GBTs relies on the efficiency of the vectors splice
acceptor and transcriptional termination signals in a given genomic context. To assess
the mutagenic efficiency of our system, we have performed qRT-PCR to quantify the
levels of wild type read-through transcript in 9 lines (Figure 4B). Expression levels were compared between three or four homozygous mutant embryos
and three wild type siblings at 5 dpf. In four out of six lines, the amount of wild
type transcript was below 1%, while one additional line had wild type transcript at
almost 4%. In the remaining four lines, the amount of wild type transcript ranged
from 9.5% to 32%.

It should be noted that lines harboring integrations into exons - st6GalNAc5tpl5, dkey-9i23.6tpl13, fam46bbtpl18 and cyp26C1tpl24 - were excluded from this qRT-PCR analysis because wild type transcript cannot be
produced in these lines. We also excluded the gene trap line flrtpl19, because it had a severe embryonic phenotype suggestive of a null phenotype (see
below). It is difficult to unequivocally interpret levels of tissue-specific specific
transcript between wild type embryos and embryos with severe developmental defects.

Assessment of mutagenicity at the phenotypic level

To test for overt homozygous phenotypes, we have in-crossed F2 or later generation
heterozygous siblings from all gene trap mutant lines. Embryos were checked for overt
phenotypes at 1 dpf, 3 dpf, 5 dpf. Two of our gene trap lines are insertional alleles
of genes with previously characterized chemically-induced mutations: nsfatpl6 and flrtpl10. All incrosses of nsfatpl6 heterozygotes resulted in approximately 25% of embryos displaying failure to inflate
the swim bladder and a progressive paralysis phenotype consistent with the previously
published phenotype of nsfast25and nsfast53[51] (Additional file 4: Figure S4A and Additional file 5: Movie 1). PCR genotyping confirmed that all paralyzed embryos with non-inflated
swim bladders were homozygous for the nsfatpl6 gene trap allele (data not shown). Similarly, all incrosses of flrtpl10 heterozygotes resulted in 25% of embryos displaying abnormal body curvature and kidney
defects similar to the phenotype of flrm477 homozygous mutants [58] (Additional file 4: Figure S4B and Additional file 6: Movie 2).

To test for postembryonic lethality, we raised GFP-positive fish from incrosses of
8 additional gene trap lines: nudCtpl3, stat5.1tpl4, st6GalNAc5tpl5, zfp36l1atpl8, nfe2I1tpl9, atp1a3atpl10, bbs7tpl11 and sgip1tpl12. For gene traps that do not affect survival of homozygotes, we expected 1/3 of adult
fish to be homozygous and 2/3 fish to be heterozygous for the gene trap. In all but
one line (atp1a3atpl10) we observed the expected Mendelian ratio of homozygous mutants versus heterozygous
fish. In the atp1a3atpl10 gene trap line, no homozygous mutants were identified after genotyping the initial
clutch of 14 GFP-positive adult survivors, even though homozygous embryos did not
display overt phenotypes at 5 dpf (Additional file 7: Figure S5). We then followed the survival rate among 86 GFP positive embryos from
4 different clutches of heterozygous in-cross over a period of one month. We discovered
that a severe drop in survival occurred between 8 dpf and 10 dpf with no further change
beyond 20 dpf. Forty five percent (42/86) of larvae survived to one month. We then
genotyped adult fish (n = 24) from two additional clutches and failed to find homozygous
mutants among them. Together with the initial genotyping data we obtained 0/38 homozygous
embryos instead of the expected ratio of approximately 13/38 (p < 0.001). This phenotype
is consistent with postembryonic lethality observed in atp1a3 mutant mice [59].

Morpholino (MO) knockdown phenotypes have been published for two mutants recovered
in our screen, atp1a3atpl10 and bbs7tpl11[60,61]. Morpholino knockdown of Atp1a3a resulted in dilated brain ventricles and electrophysiological
defects in Rohon-Beard neurons, resulting in defective touch response [61]. In incrosses of atp1a3atpl10 heterozygotes we did not observe embryos with severely dilated (or otherwise morphologically
defective) brain ventricles, but we have not assessed them for more subtle defects
(Additional file 7: Figure S5A). We also did not observe severe touch response defects reported in morpholino
injected embryos at 60 hpf. However, larval lethality of atp1a3atpl10 homozygotes clearly supports an essential role for Atp1a3a in neural development
and/or physiology.

For bbs7, Yen and colleagues observed absent or reduced Kupffer’s vesicle (KV) in 28.1% of
embryos injected with 500 μM solution of morpholino. The percentage of embryos with
KV defects was reduced to 6.7% when 250 μM solution of the same morpholino was used,
indicating that this phenotype was highly dose-dependent. We did not observe a significant
fraction of embryos with KV defects in bbs7tpl11 heterozygous incrosses (data not shown). Yen and colleagues also noted that approximately
14% of embryos injected with either 250 μM or 500 μM solution of bbs7 morpholino displayed defects in cardiac jogging: the first morphologically observable
indication of left-right patterning [60]. In our incrosses of bbs7tpl11, we observed a low percentage (<5%) of embryos with heart looping or jogging defects
or delay at 1 dpf, but a majority of these embryos had normal hearts by 3 dpf. Notably,
embryos homozygous for bbs7tpl11 retained over 9% of full-length transcript, and the level of endogenous full-length
bbs7 transcript varied 14-fold among homozygous embryos while the variation was only 2-fold
among wild type controls. Furthermore, bbs7tpl11 homozygotes are viable and fertile. Based on these observations, we believe that
bbs7tpl11 is a hypomorphic allele. Hypomorphic nature of bbs7tpl11 allele explains why we did not observe phenotypes nearly as severe as noted for high-dose
morpholino knockdown. It has also been suggested that mutations in modifier genes
are required for full penetrance of Bardet-Biedl Syndrome [62-65], making it possible that the observed bbs7 MO phenotypes may be specific to the genetic background in which they were observed.

Reversion of gene trap mutations by Cre recombinase

The internal components of the GBT-B1 vector (the gene trap cassette and the UAS:eGFP
cassette) are flanked by loxP sites in direct orientation, analogously to the arrangement
in GBT-R15 and GBT-RP2 [27,28]. Since mutagenic properties of gene breaking transposons are brought about by the
splice acceptor and polyadenylation/transcriptional termination signals, removal of
these components should lead to reversion of the insertional mutation.

To test the efficacy of gene trap excision by Cre recombinase, we crossed nsfatpl6 to Tg(UAS:mRFP)tpl2 homozygotes and injected 25 pg of in vitro transcribed Cre mRNA into the yolks of 1-cell embryos. We found that Cre recombinase
was extremely efficient at excising the gene trap cassette, as evidenced by mosaic
and nearly complete loss of both mRFP and eGFP expression in Cre-injected embryos
(Figure 6A, B). We then tested if this Cre activity is sufficient to revert mutant phenotypes
in injected embryos. We injected 75 pg of in vitro transcribed Cre mRNA into the yolks of 1-cell embryos obtained from incross of gene
trap (nsfatpl6, flrtpl10 and atp1a3atpl10) heterozygotes. For nsfatpl6 and flrtpl10, we then prepared DNA from individual non-paralyzed 5 dpf embryos with inflated swim
bladders and performed PCR genotyping for gene trap homozygocity. In both cases, we
found 5/24 genotyped embryos to be homozygous for the gene trap (data not shown).

Figure 6.Manipulation of the nsfatpl6 gene trap using Cre and Flp recombinases. Embryos containing the nsfatpl6 gene trap and UAS:mRFPtpl2 were injected with 25 pg of Cre (A, B) or 600 pg of eFlp (C, D) mRNA. Groups of four representative embryos, with the bottom embryo representing
low recombinase activity. Note simultaneous loss of GFP and RFP in panels A and B (injection of Cre RNA), and loss of GFP without loss of RFP in panels C and D (injection of eFlp RNA).

For atp1a3atpl10, we scored Cre-injected embryos for GFP and raised GFP-positive embryos to adulthood.
Adult fish were genotyped, and 3/10 were found to be homozygous for the reverted atp1a3atpl10R allele (data not shown), demonstrating efficient reversion of the mutant allele.

Removal of UAS:eGFP by Flp recombinase

Our gene trapping cassette has a built-in UAS:eGFP component, which allows instant
visualization of the trapped gene’s expression pattern. To increase versatility of
our mutant lines, we have flanked UAS:eGFP sequences by direct FRT sites. Upon expression
of Flp recombinase, recombination between the FRT sites would result in excision of
the UAS:eGFP reporter without affecting the mutagenic Gal4-VP16 component. Excision
of the eGFP reporter would make the GFP channel available for utilization in other
GFP-reliant transgenic applications. For the proof-of-principle of Flp-recombinase
functionality, we injected 600 pg of in vitro transcribed Flp recombinase RNA into
the yolks of 1-cell embryos heterozygous for the nsfatpl6 gene trap and containing Tg(UAS:mRFP)tpl2. As expected, injection of Flp mRNA did not lead to reduction of mRFP expression.
Somewhat surprisingly and in contrast to injection of Cre mRNA, a majority of the
embryos did not display a significant reduction in eGFP expression either. Only about
10-15% of the embryos displayed significant loss of eGFP (Figure 6C, D). Embryos with significantly reduced GFP expression were raised to adulthood
and six resulting adults were outcrossed to Tg(UAS:mRFP)tpl2. Germline-transmited Flp-mediated UAS:eGFP excision events were scored by complete
absence of eGFP expression in the presence of mRFP. Five out of six outcrossed adults
displayed complete loss of eGFP expression in the germline (Additional file 8: Table S2). As expected, the Flp-deleted allele of nsfatpl6 retains the ability to activate Tg(UAS:mRFP)tpl2 in trans, and 50% of embryos display mRFP expression. We conclude that UAS:eGFP cassette can
be readily removed from GBT-B1 gene trap lines.

Discussion and conclusions

The main principle underlying our insertional mutagenesis system is shared with the
recently reported 5’ gene trap vectors GBT-R14, R15 and R16 [27,28], which use highly efficient splice acceptor and polyA to disrupt endogenous transcripts
and mRFP to visualize the mutated gene’s expression pattern. We have expanded the
versatility of these gene-breaking transposons by splitting the expression-reporting
core into two separate entities. We have replaced mRFP with Gal4-VP16 in the mutagenic
entity of the vector. To report the presence of Gal4-VP16 we included a Gal4 UAS reporter
(UAS:eGFP). The UAS:eGFP component is removable by Flp recombinase for situations
where the GFP channel is needed for other purposes such as visualization of another
transgene. Replacement of mRFP with Gal4-VP16 serves a dual purpose. First, transcriptional
activator property of Gal4-VP16 amplifies the signal of the trapped gene, enabling
visualization of low level IMG expression. For example, Gal4-VP16 dependent GFP and
RFP expression was observed in the pancreas of the nsfatpl6 gene trap line – an organ which was previously not known to express nsfa. Second, while mutant lines with no phenotype have rather limited research value,
our “non-phenotypic” gene trap lines can be used as Gal4 drivers for ectopic expression
of any UAS-controlled transgene of interest. Notably, neither epigenetic silencing
of UAS:eGFP nor removal of UAS:eGFP by Flp affect the ability of our gene traps to
act as Gal4 drivers. Furthermore, the standard considerations apply when using our
gene traps as Gal4 drivers: the UAS:driven transgene may be affected by silencing
in some or all tissues, and it may or may not express in any individual cell, as illustrated
for mRFP in Figure 4. In that sense, our gene traps are not different from previously published Gal4 enhancer-
and enhancer/gene-traps [23,31,32]. We would like to note that compared to enhancer traps with hsp70 minimal promoter, our gene trap does not exhibit background expression in non-specific
tissues such as the heart and the muscle.

The use of Gal4-VP16 as the primary gene trap reporter may also raise some concerns.
It has been noted that Gal4-VP16 can be toxic when expressed at high levels [32,45-47]. We recovered several lines with very high levels of fluorescent reporter (GFP and
RFP) expression, for example tlp26, tpl37 and tpl39, and did not observe overt phenotypes in incrosses of these lines. Furthermore, there
is a significant difference in how Gal4-VP16 is expressed between different experiments.
Our gene traps produce fusion protein under the control of a single-copy endogenous
promoter, while other experiments used strong exogenous promoter constructs, potentially
in multiple copies. In our gene trap lines Gal4-VP16 is expressed as a fusion protein
with the N terminus of the protein encoded by the endogenous gene, which is not usually
the case in other experimental paradigms. Together with the observation that Gal4-FF
is inactive in our gene trap context, our data argues that the strength of Gal4-VP16
is just right for our gene trap context, and that expression levels our gene traps
achieve are unlikely to cause of toxicity.

Another potential concern for using Gal4-VP16 as the primary gene trap reporter is
that expression of UAS-controlled transgenes are susceptible to variegation and silencing
in zebrafish [47,66]. To circumvent this problem, we employ a second reporter, UAS:mRFP, to ascertain
the presence of the gene trap allele. We also tend to select high expressors when
we propagate our gene trap lines. Nonetheless, after F5 generation, we no longer observe
GFP expression in several lines including ebf3tpl16 and fnbp1tpl24. Despite of loss of GFP expression, these gene trap lines can still be used as Gal4
drivers, as they successfully transactivate UAS:mRFP. Replacing the 14XUAS with a
less repetitive variant such as nrUAS [66] may be worthwhile if sensitivity for low expression levels can be retained.

The third potential concern for using Gal4-VP16 as the reporter may be the requirement
that IMG-Gal4-VP16 fusion proteins must enter the nucleus, bind the DNA and activate
transcription. This excludes a significant subset of genes as potential targets. For
example, Gal4-VP16 fusions with proteins containing amino-terminal signal peptide
would be unable to enter the nucleus. Such proteins constitute about a fifth of vertebrate
proteomes [67]. Many other protein domains have been noted to affect protein localization within
the cell. We were concerned that use of Gal4-VP16 as the primary reporter will introduce
significant bias in intracellular functions of trapped genes, such that we would mainly
target transcription factors and other proteins which primarily function in the nucleus.
While the subset of trapped genes presented here is too small to exclude or confirm
the possibility of such bias, we note that among the trapped genes, two are components
of the SNARE complex involved in secretory pathway (nsfa and snap25), and two are involved in cilia biogenesis (bbs7 and fleer). Thus, genes involved in a variety of cellular processes can be mutated using our
vector. Furthermore, GBT-R15 and GBT-RP2 are especially suitable for trapping genes
coding for secreted proteins [28], making our Gal4-VP16-based approach complementary.

It would be possible to expand the repertoire of genes amenable to trapping using
Gal4-VP16 (e.g. eliminate bias against proteins with N-terminal signal sequence) by
using viral P2A/T2A co-translation systems demonstrated to work in zebrafish [68,69]. We do not favor this strategy, since there appears to be a positive side effect
of using Gal4-VP16 as the primary gene trap reporter. Among gene trap lines we characterized,
six have integrations into intron 1, and two additional lines have integrations into
exon 2, which effectively results in fusion transcript with exon 1 (Figure 4A). Gene traps using fluorescent reporters do not appear to have a similar 5’ bias
[22,27,28,70,71]. The 5’ bias of our vector is likely brought about by the requirement that IMG-Gal4-VP16
fusion protein must enter the nucleus, bind DNA and activate transcription of eGFP
and mRFP under the control of Gal4 UAS. One or more of these functions may be compromised
by addition of a large polypeptide with one or more functional domains. As a consequence
of this 5’ gene trap bias, a shorter part of the endogenous gene is expressed, which
increases the likelihood of null alleles. Inclusion of P2A/T2A would eliminate this
beneficial bias of Gal4-VP16 gene traps toward 5’ ends of genes.

Efficient mutagenic potential of our vector is observed at both molecular and phenotypic
level. Among the 25 molecularly characterized lines, four are integration into exons
and therefore null alleles. Almost half the lines analyzed by RT-PCR (4/9) display
wild type transcript levels below one percent and therefore should be considered null
alleles as well. A fifth has wild type transcript level below five percent, making
it a possible null mutant, too. The flrtpl10 line was not analyzed by RT-PCR due to developmental abnormalities but displays a
homozygous phenotype very similar or identical to that of the corresponding chemically-induced
null mutant. Thus, it is safe to assume that well over 50% of mutants recovered with
GBT-B1 will be null alleles. How does it compare to other published insertional mutagenesis
systems? Assessment of mutagenicity of the only insertional mutant used in large scale
in zebrafish so far, the pseudotyped retrovirus, demonstrated that 4/10 integrations
into intron 1 resulted in transcript levels below 5%, while 1/8 integrations immediately
upstream transcription start site and 0/5 integrations into other introns produced
likely null alleles [72]. A more appropriate comparison would be with other transposon-based insertional mutagenesis
systems. Unfortunately, mutagenicity of most other transposon systems was not systematically
assessed at the molecular level [22,32,71]. Recently published Flex-based vectors reduced the levels of endogenous transcript
to just below 7% [70], which is easily surpassed by our vector. It is also interesting to note that GBT-B1
appears to be more mutagenic than the parental vector GBT-R15 [27,28]. This may be caused by the addition of the UAS:eGFP cassette. Even though this cassette
is in antisense orientation in the trap, the SV40 p(A) used to terminate transcription
of eGFP is bidirectional [73]. Even though SV40 p(A) is not efficient enough to cause mutations on its own [26], it may contribute to reduction in endogenous transcript. Furthermore, several potential
splice acceptor sites can be identified in the antisense strand of 14XUAS:eGFP cassette
(http://wangcomputing.com/assp/index.htmlwebcite[74]). It is also possible that replacement of ^mRFP with ^Gal4-VP16 added an exonic splice
enhancer or removed an exonic splicing silencer [25], thus improving the efficiency of the short carp β-actin splice acceptor used in
both vectors. Each of these factors may account for the minor increase in mutagenicity
of GBT-B1 compared to GBT-R15. However, mutagenicity of GBT-B1 is clearly lower than
that of GBT-RP2 [28]. GBT-RP2 contains an additional carp β-actin splice acceptor in the 3’ gene trap
component of the vector, which may contribute to higher mutagenicity of this vector.
GBT-RP2 vector also uses a poly (A) which is derived from ocean pout antifreeze gene
and is thought to contain a potential boundary element [75]. It would be interesting to test if replacement of zebrafish β-actin p(A) with ocean
pout antifreeze p(A) would increase the mutagenicity of our vector to RP2 knockdown
levels, or if an additional splice site would still be required.

While qPCR provides the precise degree of gene inactivation at the molecular level,
the level of disruption needed to achieve a loss-of-function phenotype is likely to
be different from gene to gene. Loss-of-function chemically-induced mutants have been
previously described for two of the genes mutated in our screen, nsfatpl6, flrtpl10. In both cases, the phenotypes of our insertional mutants appear to be indistinguishable
from corresponding chemically-induced alleles [51,58]. Interestingly, gene trap integrations in these two genes have occurred into the
first intron. Only 4/744 and 18/651 amino acids are retained by Nsfa- and Fleer-Gal4-VP16
fusion proteins, respectively.

Our mutagenesis system offers an ability to conditionally revert mutagenic insertions
into non-mutagenic ones by Cre-recombinase mediated excision of the Gal-VP16 (and
UAS:eGFP) sequences. Reversion of mutations by injection of Cre mRNA is very useful
in determining causal relationship between gene trap integrations and observed phenotypes.
Tissue- and/or time-specific reversion of the mutation attained by breeding gene trap
mutants to lines expressing CreER or CreERT2 in a tissue-specific manner would enable
dissection of spatiotemporal requirement of the trapped gene. This functionality gives
mutant alleles made with GBT-B1 an advantage over alleles made chemically, with various
targeted nucleases, or with retrovirus. However, it would be even more beneficial
to combine the high mutagenicity of GBT-B1 (perhaps improved to GBT-RP2 levels) with
the full conditionality offered by two-recombinase inversion systems such as FleX,
recently adapted for use in zebrafish [70,71]. Combination of additional functionality offered by Gal4-VP16 with high mutagenicity
and full conditionality would provide a very powerful tool for dissection of gene
function in the zebrafish.

Transgenesis procedures

Gene trap construct GBT-B1 (pDB783) was purified using Qiagen miniprep protocol including
the optional PB wash step. Plasmid DNA (25 pg) was co-injected with 25 pg of Tol2
RNA into 1-cell zebrafish embryos in standard Tol2 transgenesis protocol [37,77]. Embryos injected with GBT-B1 were screened on Zeiss Axioscope or Zeiss Axioimager
(both with 5X Fluar objective) for high levels of GFP fluorescence at 3 dpf. Approximately
20-30% of embryos surviving to day 3 were considered high expressors and raised to
adulthood. The same methods were applied to generate the Tg(Tol2/14XUAS:mRFP) line, embryos were injected with pTol2/14XUAS:mRFP (pDB788) with Tol2 transposase
mRNA and raised to adulthood. Adults were incrossed and embryos were screened for
leaky expression of mRFP. A single transgenic line was established. To produce Tg(Tol2/gCry:GFP,14XUAS:mRFP)tpl2 line, we injected embryos with Tol2 mRNA and Tol2/gCry:GFP, 14XUAS:mRFP (pDB790),
screened for GFP expression in the lens and raised to adulthood. Adults were incrossed
and embryos were screened for lens-specific GFP expression. A single transgenic line
was established.

Inverse PCR

Genomic DNA was prepared from batches of 20 GFP positive and GFP-negative embryos
from an F1 or F2 outcross. In separate reactions, the genomic DNA was digested with
NlaIII, TaqI, NheI/SpeI/XbaI/XmaJI or BamHI/BclI/BglII, then diluted and ligated overnight
as described in [28,78]. Several different primer combinations were used in inverse PCR. For most lines,
the first PCR reaction was carried out using primers Tol2-F13 and Tol2-R4 (primer
sequences are listed in Additional file 9: Table S1). 0.1-1uL of the first PCR was used to carry out the second PCR reaction
using primers Tol2-F11 and Tol2-R5. To identify genes mutated in tpl17, tpl23 and tpl25, two sets of inverse PCR reactions were carried out. For 5’ end of the gene trap,
first PCR was carried out with Tol2-F8 and B1/5’No3 and the second PCR was carried
out with Tol2-F4 and B1/5’No2. For 3’ end of the trap, the first PCR was carried out
with Tol2-R3 and B1/3’No1 and the second PCR was carried out with Tol2-R4 and B1/3’No2.

Identification of Gal4 fusion transcripts by 5’RACE

Total RNA was prepared from a pool of 20 GFP positive and a pool of 20 GFP negative
5 dpf embryos using Absolutely RNA miniprep kit (Agilent Technologies, Cat# 400800).
cDNA was made from 250-500 ng of total RNA using SuperScript™ II Reverse Transcriptase
(Invitrogen, Cat # 18064–022) following previously described protocol [28,79]. Race-ready cDNA was amplified by PCR using primer mix containing KJC-002 and KJC-003
at a ratio of 1:5 and Gal4-R2. Second PCR reaction was performed using either 1 μL
of undiluted or 1 μL of 1:10 dilution of the first PCR, using KJC-004 and Gal4-R3
primers. PCR products from the second PCR reaction were run on 1% agarose gel, and
bands obtained on GFP-positive embryo DNA but absent from GFP-negative siblings were
purified using either Qiagen or Thermofisher Fermentas Gel Extraction kits and sequenced.

Confirmation of Gal4 mRNA fusion by RT-PCR

Once gene trap integration linked to GFP expression was identified by iPCR (or 5’RACE),
the presence of Gal4 mRNA fusion was confirmed by RT-PCR. For this, cDNA was made
from 250-500ng of total RNA prepared from a pool of GFP positive 5 dpf embryos using
SuperScript™ III Reverse Transcriptase (Invitrogen, Cat # 18080–044) and random hexamers
following protocol supplied by the manufacturer. 1 μl of the cDNA was used for PCR
amplification using forward genomic primer for an exon upstream of the integration
and a reverse primer for Gal4 (Gal4-R2 or Gal4-R3). PCR bands were sequenced with
Gal4 reverse primer to confirm correct reading frame of the fusion mRNA.

For tpl6 and tpl10 lines, additional Southern analyses were performed on genomic DNA digested with HindIII,
NsiI and HindIII/NsiI.

Genotyping

Genotyping strategy was designed for gene trap insertions with known genomic integration
sites. Genotyping was typically performed using three-primer PCR: forward genomic
primer upstream of the integration, reverse genomic primer downstream of the integration,
and a gene trap (Tol2) primer. Wild type allele produces an amplicon between two genomic
primers, while the gene trap allele gives rise to a product between the gene trap
primer and one of the genomic primers. Primers were selected so that wild type band
and gene trap band would differ in size. For an example, see Additional file 7: Figure S5, lanes 17 and 18. Primer sequences are listed in Additional file 9: Table S1, and exact genotyping PCR conditions are available upon request. In cases
where three-primer PCR failed, standard two primer PCR was performed using one transposon
primer and one genomic primer on gene trap positive and gene trap negative embryo
DNA.

RT-qPCR

RT-qPCR was used to determine the efficiency of mRNA knockdown of insertionally mutated
gene by quantifying relative amount of intact mRNA present in homozygous mutants versus
wild type siblings. DNA and RNA were isolated from 3 GFP-negative and 10–15 GFP-positive
5 dpf embryos using Trizol reagent (Ambion). DNA fraction was used for genotyping,
and RNA was used to prepare cDNA from 3 wild type and 3–4 homozygous mutant siblings.
qPCR was carried out using LightCycler® 480 SYBR Green I Master kit (Roche, Cat #
04707516001) with genomic primers for trapped gene’s exons flanking the transposon
integration. Each sample was analyzed in triplicates. β-actin was used as a “reference”
gene to normalize for differences in cDNA yields. PCR conditions were as follows:
5 min initial incubation at 95°C, followed by 42 cycles of 95°C for 10 sec, 57°C for
15 sec and 72°C for 1 min, and ending with a final step at 72°C for 7 min. qPCR data
were analyzed using LightCycler® 480 1.5 software. Expression levels of intact mRNA
of the mutated gene (”target” gene) for each sample were calculated as ratio of CT
values for “target” and “reference” genes, averaged over triplicates assuming equal
PCR amplification efficiencies (=normalized expression) [80]. Genotyping and qPCR primers are listed in Additional file 9: Table S1.

In situ hybridization

Analysis of co-expression of endogenous nsfa transcript with eGFP was performed by whole-mount in situ hybridization on 1 dpf embryos as described previously [50]. nsfa full-length cDNA was obtained by PCR amplification using primers nsf/kpn-F1 and nsf/Cla-R1
and 5 dpf embryo cDNA as the template. The 2.2 kb PCR band was cloned into the pCR®II-TOPO®
vector (Invitrogen K4600-01). Resulting plasmid was linearized with BamHI and riboprobe
synthesis was performed using DIG RNA Labeling kit (Roche 11 175025910) with T7 RNA
polymerase in 20 μL of reaction volume. The reaction was carried out at 37°C for 2
h, and incubated for additional 15 min with 2 μl of DNase I at the same temperature.
RNA was precipitated by adding 2 μL of 7.5 M Lithium Chloride and 75 μl of 100% ethanol
following incubation at −20°C overnight. Following centrifugation, RNA pellets were
washed with 70% ice-cold ethanol and dissolved in 30 μl of RNase-free water. eGFP
probe was obtained by PCR amplification with ^eGFP-F1 and Cla/BGYFP-R1 (primer sequences
are in Additional file 9: Table S1) using GBT-B1/pDB783 as a template. Probes for ebf3, cyp26c1 and snap25b were PCR amplified from 5 dpf embryo cDNA using corresponding primers listed in Additional
file 1: Table S1. All subsequent steps were the same as described for nsfa probe.

Site-specific recombinases

Cre mRNA was transcribed using pT3TS/Cre (pDB638) as the template as described in
[27,28]. For reversion of gene trap mutations, 25–75 pg of in vitro transcribed Cre mRNA was injected into 1-cell zebrafish embryos.

To synthesize Flp RNA, we first amplified eFLP coding sequence using primers Bgl-Flp-F1
and XbaCla-Flp-R1, with pCMV-Flpx9.MiDB [81] as the template. PCR product was cloned into pJet1.2 (ThermoFisher Fermentas) and
then subcloned into pT3TS [82] to generate pDC31. pDC31 was linearized with BamHI and transcribed using mMessage
Machine T3 in vitro transcription kit (Ambion). 600 pg of the in vitro transcribed eFLP mRNA was injected into the yolks of 1-cell stage zebrafish embryos
in 3 nL volume.

Acknowledgements

We thank Bashar Kako for technical assistance cloning RT-PCR fragments for in situ hybridization. We thank Ryan Gill and zebrafish facility staff for fish care. Our
research was supported by startup funds provided by Temple University to DB and NIH
Grant R01-HD061749 to DB and JB.